Synaptic strength is subject to activity-dependent changes over periods of milliseconds to minutes, a phenomenon referred to as short-term synaptic plasticity (STSP). STSP has a direct influence on computations performed by neural circuits and must be understood to fully understand brain function. The synaptic environment is subject to significant activity-dependent pH fluctuations but their impact on the pH-sensitive mechanisms underlying neurotransmission is rarely considered despite their likely influence of multiple mechanisms underlying STSP. We have developed fluorescent genetically-encoded pH indicators allowing single action potential resolution of pH dynamics in the synaptic cleft of the Drosophila NMJ. Our preliminary data reveal the surprising extent to which the cleft alkalinizes (see preliminary data) and it is highly likely that this also happens at vertebrate synapses that employ the Ca2+/H+ exchanging plasmamembrane Ca2+-ATPase (PMCA). Furthermore, our preliminary data point to cleft alkalinization potentiating both quantal size and Ca2+ entry during burst firing. Our long-term goal is to elucidate the means by which pH fluctuations are incorporated into STSP mechanisms. Within this proposal we will examine the hypothesis that activity-dependent cleft alkalinization has been incorporated into gain mechanisms that sustain neurotransmission during burst firing. Using molecular genetic techniques, electrophysiology and fluorescence imaging we will test our working hypotheses that presynaptic voltage-gated Ca2+ channels (VGCCs) and postsynaptic ionotrophic glutamate receptors (iGluRs) are potentiated by alkalinization at their extracellular faces in the cleft. Our Research Strategy is broken down into three separate aims: Aim 1: Elucidate the influence of synaptic cleft alkalinization on presynaptic Ca2+ entry during bursts. Aim 2: Elucidate the mechanisms by which synaptic cleft alkalinization affects quantal size during bursts. Aim 3: Investigate the impact of neurotransmitter release on cleft pH change at individual active zones. Here we develop a test bed for investigating the contribution of activity-dependent pH fluctuations to mechanisms underlying STSP. Beyond their immediate employment in addressing the aims above, the reagents we develop will be useful for subsequent investigations into the contribution of pH-sensitive STSP mechanisms to circuit function and behavior in Drosophila, potentially providing insight into neurological disorders with an acid-base imbalance component such as seizure disorders and certain intellectual disabilities.